I did a normal single marker PCR for my SSR markers and analysed the PCR product on a 3% agarose gel. two of the markers show two bands on the gel, the bands are very close together but there is no size overlap as these are distinct. one is the expected band and it is very clear but the second band which is slightly bigger is faint even though it is still visible. I only see this on two markers only. What could this be? is it a second allele maybe or my primers might amplify another marker that is close to my target marker?
Perhaps your amplimer has a polymorphism in it and the pcr process melts the products and produces normal/normal and also normal/polymorphic product which has a bubble at the polymorphism so runs slower than the n/n product so looks larger. At the right temperature heteroduplexes can run at a different size from the normal product
I think, you have a stutter band. This is the feature of your Taqpol. Looks like these guyes observed similar effect.
http://mbrc.shirazu.ac.ir/article_3789_10b7d720b5b93b199c8c2fc912c351fc.pdf
It is ok. Usually its concentration should not exceed 10-15% of the main band.
In "Faint Band" case, I think you have to make sure from the followings with same order:
1. DNA carry over (Negative Control)
2. Primer dimer (Negative Control)
3. Temperature (Optimization)
you have an heterozygous organism, diploid.
your results are ok.
Heterozygosity does not lool like shadow. In some cases, excluding statters it may be a result of chimerism.
Thanks for all the interesting answers, I am looking into each case, and thank you for the article recomendation. I will definitely consider each answer as I continue to figure this out.
I think your primer amplified more lilkely similar sequence in your sample found in different region of the same chromosome or other chromosomes. You might check the specificity of your primer to your target region using BLAST.
you can see http://www.cdfd.org.in/SILKSAT/ssragrs.html and http://www.cdfd.org.in/SILKSAT/index.php?f=protocol_ssr and read Mason 2014. SSR genotyping.
this link is useful
https://www.researchgate.net/post/Double_bands_after_PCR
regards
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